Influence of maternal psychological distress during COVID-19 pandemic on placental morphometry and texture

The Coronavirus Disease 2019 (COVID-19) pandemic has been accompanied by increased prenatal maternal distress (PMD). PMD is associated with adverse pregnancy outcomes which may be mediated by the placenta. However, the potential impact of the pandemic on in vivo placental development remains unknown. To examine the impact of the pandemic and PMD on in vivo structural placental development using advanced magnetic resonance imaging (MRI), acquired anatomic images of the placenta from 63 pregnant women without known COVID-19 exposure during the pandemic and 165 pre-pandemic controls. Measures of placental morphometry and texture were extracted. PMD was determined from validated questionnaires. Generalized estimating equations were utilized to compare differences in PMD placental features between COVID-era and pre-pandemic cohorts. Maternal stress and depression scores were significantly higher in the pandemic cohort. Placental volume, thickness, gray level kurtosis, skewness and run length non-uniformity were increased in the pandemic cohort, while placental elongation, mean gray level and long run emphasis were decreased. PMD was a mediator of the association between pandemic status and placental features. Altered in vivo placental structure during the pandemic suggests an underappreciated link between disturbances in maternal environment and perturbed placental development. The long-term impact on offspring is currently under investigation.

MRI data acquisitions. Axial single shot fast spin echo T2-weighted images were acquired on a 1.5 Tesla Discovery MR450 scanner (GE Healthcare, Milwaukee, WI) using an eight-channel surface receiver coil. Acquisition parameters were as follows: echo time = 160 ms, repetition time = 1100 ms, field of view = 420 × 420 mm, and slice thickness = 4 mm. The final in-plane resolution was 1.64 × 1.64 mm with 40-60 consecutive slices for full placental coverage. Neither sedation nor contrast was used during MRI studies. Each subject was scanned up to two time points in the fetal period.
MRI data post-processing. Placentas were manually segmented in the plane of acquisition and then corrected on the other planes to ensure spatial consistency using ITK-SNAP ( Fig. 1) 38 . The segmentations were performed by two research engineers (K.K., N.R.A.) with 3-5 years of experience in MRI placental segmentation and were reviewed by a senior neonatologist (N.A.) with more than 7 years' experience. Intra-rater and inter-rater reliability of the placenta segmentations were determined using 30% of randomly selected scans (80 pre-pandemic and 27 pandemic) by the two trained raters (K.K., N.R.A.). Intra-and inter-rater reliabilities using intra-class correlation coefficient were all higher than 0.97 for both pre-pandemic and pandemic cohorts. All were blinded to pandemic vs. pre-pandemic cohort.

Shape features.
To characterize three-dimensional (3-D) placental shape, three shape features were used: volume, thickness, and elongation. Detailed elaborations of the determination of these three features have been provided in Dahdouh and Andescavage's studies 22,24 , and brief descriptions of the features are summarized here. The volume was calculated based on the triangular meshes of the 3-D placental model 22 . Thickness was defined as the maximal distance between the points of the placenta belonging to the maternal surface and their projection on the fetal surface 22 . Elongation was defined as the length of the longest branch of the 3-D medial axis skeleton of the shape 22 . All calculations were implemented in MATLAB R2019a (The MathWorks, Inc., Natick, MA).

Statistical analyses.
Univariate analyses were performed to explore the demographic data. The Kolmogorov-Smirnov test was first utilized to test the normality of the continuous variables, including GA; maternal age; maternal weight at MRI; and birthweight, and the results showed that all these variables were not normally distributed. The fetal and maternal demographics were therefore compared between pre-pandemic and pandemic cohorts using Wilcoxon-Mann-Whitney tests for GA, maternal age, maternal weight at MRI and birthweight and using Chi-square tests for fetal sex, number of scans, maternal parity, maternal race/ethnicity and the temporal distribution of subject recruitment.
The generalized estimating equation (GEE) was utilized to determine the following associations for comparing the pre-pandemic and pandemic cohorts 43 . First, the associations between pandemic status (pre-pandemic: 0; pandemic: 1) and maternal distress measures (SSAI, STAI, PSS and EPDS) were determined using GEE, adjusting for GA at MRI (weeks), given the known effects of GA on placental MRI features. Second, the associations between pandemic status and placental shape features were analyzed using GEE. The primary independent variable was the pandemic status (pre-pandemic: 0; pandemic: 1), and all GEEs were adjusted for GA at scan (weeks). Third, the associations between pandemic status and placental textural features were analyzed using GEE, adjusting for GA at scan (weeks). In addition, we further adjusted maternal distress (low distress: 0; high distress: 1) in the GEEs to determine whether maternal distress is a mediator in the association between pandemic status and placental shape and textural features. High distress was defined as any one of the four distress measure summary scores being greater than their corresponding threshold (SSAI: 40; STAI: 40; PSS: 15; EPDS: 10) 32,34,36,37 . Furthermore, the association between placental features and birth weight (g) by pandemic status were also investigated using GEEs, adjusting for GA at MRI (weeks), maternal distress (0: low distress, 1: high distress,) and GA at birth (weeks). Mediation analyses were further implemented to determine whether prenatal maternal distress works as a mediator on pandemic status and placental features 44 . Three steps of mediation analyses were conducted using GEE: (1) the association between placental features and the pandemic status, adjusting for GA www.nature.com/scientificreports/ at MRI; (2) the association between maternal distress measures and the pandemic status, adjusting for GA at MRI; (3) the association between placental features and the pandemic status, adjusting for GA at MRI and the significant maternal distress measures found in the previous step. Sensitivity analyses were conducted on two additional covariates, fetal sex and maternal weight, and their effect on the placental features were examined. Lastly, the time trend was investigated by fitting two GEE models (one before the pandemic and the other during the pandemic) to explore the associations between placental features and date of evaluation, adjusting for gestational age at MRI (weeks). The time trend throughout the study period was fitted using nonlinear mixed-effects estimation with the quadratic spline function 45 . The critical value for statistical significance was set as 0.05. The q-values calculated by the false discovery rate method for the number of features in each set (3 for shape features; 4 for the first set, 6 for the second set and 10 for the third set of textural features) were also reported to reflect significant parameters under multiple comparisons 46 . The unstructured correlation matrix was utilized in all GEE models, with the robust sandwich covariance matrix 47 . All analyses performed in this study were conducted using MATLAB R2019a (The MathWorks, Inc., Natick, MA, USA), and all hypothesis tests were 2-sided.

Results
Demographics. Participant recruitment is shown in Fig. 2

Placenta shape features and pandemic status.
Our data showed that the pandemic cohort had higher placental volume (least squares mean: 637.9 vs. 594.0 cm 3 , p = 0.02) and placental thickness (least squares mean: 5.3 vs. 4.9 cm, p < 0.01) when adjusting for GA at MRI in the GEE models ( Supplementary Fig. 1). In contrast, placental elongation was reduced in the pandemic cohort (least squares mean: 17.0 vs. 17.8 cm, p = 0.01) (Supplementary Fig. 1). After further adjusting for maternal distress (low vs. high distress), association of increased placental volume and thickness in the pandemic cohort remained unchanged (Table 3).  Table 3. In the first set of textural features, mean GL was reduced while kurtosis GL and skewness GL were increased in the pandemic cohort when adjusting for GA at MRI in the GEE models ( Supplementary Fig. 2). In the second set, energy was decreased in the pandemic cohort ( Supplementary Fig. 3). In the third set, long run emphasis, low GL run emphasis, high GL run emphasis, short run low GL emphasis, short run high GL emphasis, and long run high GL emphasis were all decreased, while run length non-uniformity was increased in the pandemic cohort (Sup- Figure 2. Flow diagram summarizing our subject recruitment in this study. Eligible women were recruited from community maternal fetal medicine offices and referred by their obstetrics providers. The pre-pandemic cohort was recruited between March 2014 and February 2020, and the pandemic cohort was recruited between June 2020 to April 2021. The study team spoke to potential participants alongside the obstetricians and would follow up with those interested in participation. Written informed consent was obtained from all participants before completing study procedures. www.nature.com/scientificreports/ plementary Fig. 4). After further adjusting for maternal distress (low vs. high distress), the pandemic status was associated with the above textural features except long run emphasis and long run high GL emphasis. (Table 3).

Placental features and birth weight.
The associations between placental features and birth weight by pandemic status are shown in Table 4. The placental volume, elongation and run length non-uniformity were positively associated with birth weight for both pre-pandemic and pandemic cohorts. In the pre-pandemic cohort, birth weight increased when cluster shade decreased while GL non-uniformity increased. In the pan- Apgar score at 1 minute e 8 [8,9] 8 [8,9] 8 [8,9] 0.91 Apgar score at 5 minutes f 9 [9,9] 9 [9,9] 9 [9,9]   www.nature.com/scientificreports/ demic cohort, mean GL was negatively associated with birth weight while kurtosis GL was positively associated with birth weight.

Mediation analyses. The mediation analyses show that in
Step 1, volume, thickness, elongation, mean GL, kurtosis GL, skewness GL, energy, run length non-uniformity, high GL run emphasis, short run low GL emphasis, and short run high GL emphasis were significantly changed in the pandemic cohort after adjusting for multiple comparisons (Supplementary Table 1). In Step 2, higher PSS and EPDS values were identified in the pandemic cohort as mentioned above (Table 2). In Step 3, the significance of energy, long run emphasis, and long run high GL emphasis was altered for PSS (Supplementary Table 2), and the significance of elongation, energy, long run emphasis, and long run high GL emphasis was altered for EPDS (Supplementary Table 3). These results indicate that PSS and EPDS may mediate the association between targeted placental features and the pandemic status (Fig. 3).

Sensitivity analyses.
We conducted sensitivity analyses with additional covariates including fetal sex and maternal weight at MRI in the GEE models to evaluate the association between placental features and pandemic status. The results show that fetal sex was not a significant factor on placental shape/textural features (Supplementary Table 4). While we observed that maternal weight was significantly associated with several textural features, including mean GL, variance GL, kurtosis GL, cluster shade and cluster prominence, these findings did not change the significance of the main findings after adjusting for multiple comparisons (Supplementary Table 5).
Time trend of placental features. We further conducted 2 GEE models (one for each cohort) to explore the association between placental features and scan date, adjusting for gestational age at MRI (weeks). The results show that within the pre-pandemic cohort, placental volume, thickness, elongation, GL non-uniformity, low GL run emphasis and short run low GL emphasis significantly increased over time. However, within the pandemic cohort, we did not identify a significant difference of placental features over time after adjusting for multiple comparisons (Supplementary Table 6). To better explore the temporal trends across both cohorts for the entire study period, the time trend was fitted using nonlinear mixed-effects estimation with the quadratic spline func- www.nature.com/scientificreports/ tion. The results show that placenta thickness, kurtosis GL, and skewness GL were significantly increased while elongation, mean GL, low GL run emphasis, high GL run emphasis, short run low GL emphasis, short run high GL emphasis, long run low GL emphasis and long run high GL emphasis were significantly decreased as scan date increased, after adjusting for multiple comparisons (Supplementary Table 7), consistent with the findings in Table 3.

Discussion
Our study aimed to assess changes in morphometric and textural features of in vivo placentas in women pregnant prior to and during the COVID-19 pandemic. We found that multiple features of placental morphometry and texture differed significantly between the two cohorts, mediated in part by the elevated maternal stress and depression identified in the pandemic cohort. Specifically, increased placental volume and thickness were observed amid the COVID-19 pandemic, and textural analyses showed asymmetry of image signal intensity (i.e., increased skewness GL and kurtosis GL) in the pandemic cohort. We also found larger inhomogeneous area (i.e., decreased long run emphasis) and higher non-uniformity (i.e., lower energy and increased run length non-uniformity) of placental images in the pandemic cohort. Furthermore, there were no significant temporal differences in placental development within the pandemic cohort for the duration of the pandemic period studied. Lastly, we report that the relationships between placental morphometry and birthweight differed in the pandemic and pre-pandemic cohorts, with stronger associations between placental volume, elongation, run length non-uniformity and birthweight during the pandemic.
Maternal mental health and placental adaptations. Placental adaptations to perturbations in the maternal environment and mental health can trigger adverse fetal programming 48 . Studies have shown that maternal mental health disorders and prenatal stress can alter fetal development, affecting the child's health long after the original insult such as difficult temperament, dysregulated sleep and harder-to-soothe infants, and later, lower cognitive performance and worse school achievement 49,50 . Prenatal stress has been associated with increased risk of depression, anxiety, attention-deficit/hyperactivity disorder (ADHD), conduct disorders, autism and schizophrenia [51][52][53][54] . Prior studies also have indicated the increased concerns of psychological www.nature.com/scientificreports/ disorders in pregnant women during the pandemic compared to non-pregnant women during the pandemic and compared to pre-pandemic pregnant women 55,56 , which may represent risks for offspring neurodevelopment including delayed cognitive, language, and motor development [57][58][59][60][61] . Our data showed that maternal stress and depression were elevated in the pandemic cohort, and maternal distress was a mediator of the association between pandemic status and placental morphometry and texture. In addition to detecting larger placentas in the pandemic cohort, we also found that the relationship between in vivo placental size and infant birthweight was greater when compared to pre-pandemic controls. These results build upon previous work showing that elevated maternal psychosocial stress is associated with increased placental weight at birth on evaluation of gross pathology specimen 62 . These findings demonstrated an important link between maternal mental health and the placental development, which may further influence neurodevelopmental outcomes 63-65 . Morphometric changes of the placenta during the pandemic. In this work, we show that the pandemic cohort had morphometric changes in the placenta that represent enlarged, globular placentas. While the mechanisms of increased placental volume and thickness are unclear, eccrinology and molecular studies suggest that epigenetic changes resulting from maternal distress may upregulate growth factors, resulting in increased placental size. Several potential mediators linking maternal stress and placental growth have been proposed. One such mechanism is that increased maternal stress could result in increased production of insulin-like growth factors, which can increase placental volume 66,67 . Secondly, cytokines are potential mediator between stress and placental volume. Psychological stress is linked to lower interleukin-10 (IL-10) during pregnancy 68 . Animal models with IL-10 deficiency showed an increase of up to 28% in placental size 69 . Epigenetic mechanisms further explain how maternal stress may affect specific placental gene expression patterns. In an animal study, an insulin-like growth factor called peroxisome proliferator-activated receptor alpha (PPARα), which binds tightly with protein 1 (IGFBP-1), hypoxia-inducible factor 3a (HIF3), and glucose transporter 4 (GLUT4), has increased expression for male offspring of pregnant mice with heightened prenatal stress 70 . Building on the existing data from previous pathology and molecular studies, in this study, we showed a similar pattern of accelerated in vivo placental volume during the COVID-19 pandemic. Further studies exploring these relationships are warranted. An enlarged placenta has been identified as an important factor in altered maternal-fetal nutrient supply and resulting fetal programming 71 . Khalife et al. reported significant positive associations between placental size (weight, surface area, and placental-to-birth-weight ratio) and mental health problems in boys at 8 and 16 years of age. Specifically, increased placental weight was linked with overall probable psychiatric disturbance, antisocial behavior and ADHD symptoms 71 . Abnormally enlarged placentas with altered shape also have been associated with certain medical conditions, such as maternal anemia, hypertension and diabetes [72][73][74] .
Our data show that placental volume and elongation was positively associated with birth weight in both cohorts, and this finding is in line with several previous studies [75][76][77][78][79][80][81] . We also showed that the ratio of the placental volume/elongation relative to birth weight was smaller in the pandemic vs. the pre-pandemic cohort Figure 3. The relationship between the COVID-19 pandemic, maternal mental distress and placental development. In Step 1, COVID-19 was associated with 14 placental features, including placental morphometry (volume, thickness, elongation) and texture (mean gray level (GL), kurtosis, skewness, energy, long run emphasis (LRE), run-length non-uniformity (RLNU), Low GL run emphasis (LGLRE), high GL run emphasis (HGLRE), short run low-and high-gray level emphases (SRLGLE/SRHGLE) and long run high gray level emphasis (LRHGLE). In Step 2, COVID-19 was associated with significant increase in maternal stress and depression, compared to pre-pandemic controls. In Step 3, we re-evaluate the association between COVID-19 and placental developing while adjusting for maternal distress and found that placental volume, thickness, mean GL, kurtosis, skewness, energy, RLNU, LGLRE, HGLRE, SRGLRE and SRHLE remained significantly different between pandemic and pre-pandemic controls. Features denoted by ** (q < 0.05) highlight features that remain significant after adjusting for multiple comparisons. All steps also were adjusted for gestational age at MRI. www.nature.com/scientificreports/ (i.e., the inverse of β in Table 4). This reduced placental-weight-to-birth-weight (PW:BW) ratio in the setting of the COVID-19 pandemic may be associated with adverse pregnancy outcomes that result from placental insufficiency 81 . Studies have shown that a PW:BW ratio below the 10th percentile was associated with fetal distress 81 , while small-for -gestational-age infants demonstrated an elevated birth-wight-to-placental-weight ratio 82 . In addition, the highest quintile of birth weight to placental weight ratio was associated with higher uterine artery Doppler mean pulsatility index and umbilical artery Doppler pulsatility index later in gestation 23 . Major congenital anomalies have been linked to higher (> 90th) percentiles of birth-weight-to-placental-weight ratio 83 . The impact of altered placental morphometry during pandemic on infant neurodevelopmental outcomes merits further investigation.
Textural changes of the placenta during the pandemic. We found that several textural features were significantly different in the pandemic cohort, namely features associated with asymmetry of gray scale and image heterogeneity. Specifically, we noted an increase in heterogeneity and asymmetry of image intensity of placentas in the pandemic cohort. Textural analyses of sonographic and MR images of the placenta have shown increased heterogeneity with advancing GA, a reflection of the increasing complexity of placental microstructure during gestation 24,84,85 . Regional changes in placental texture have been observed in placenta accrete [86][87][88] , while global changes in placental texture have been observed in fetal growth restriction (FGR) 24 . In this work, we found a decrease in mean GL and SRLGL along with increased kurtosis and skewness in the pandemic cohort, the converse of findings previously reported in FGR 24 . We also report an increase in RLNU during the pandemic, similar to changes reported in FGR, along with previously undescribed decreases in LGLRE, HGLRE and SRHGLE. The precise mechanisms of the microstructural changes of placentas observed from textural analyses in the pandemic cohort remain largely unclear and may in fact be multi-factorial. Previous studies have collectively pointed toward maternal psychosocial stress pathways that may alter the epigenetic signature in placentas [89][90][91][92][93][94] . First, prenatal maternal stress is linked with higher levels of maternal cortisol 89 , and the increase in maternal cortisol alters uteroplacental metabolites such as serotonin 90,95 , which is associated with beta cell heterogeneity in tryptophan hydroxylase protein induction during pregnancy 91 . Second, differential expression of placental glucocorticoid receptors has been reported in the presence of elevated maternal stress 92,93 , and a study has shown that glucocorticoid treatment could lead to increased glucocorticoid receptor mRNA variants detected in the human placenta, where GR-α and GR-1C mRNA having the highest expression 96 . Third, placental DNA methylation changes are associated with increased exposure to maternal stress 94,97,98 . These DNA methylation changes could regulate placental-specific gene expression, including monoallelic expression and X-chromosome inactivation in the placenta, leading to the changes of placental context 94,97,98 . These alterations in placental metabolism and gene expression associated with maternal distress may result in microstructural changes that can be detected by texture analyses and deserve further clinical validation particularly for the pandemic population.
Our data show that altered GL measures, namely decreased mean GL and increased kurtosis GL, of the placenta were associated with increased birth weight in the pandemic cohort, which may reflect altered placental microstructure; while the etiology of these changes is not fully elucidated, these may be related to pandemic related physiologic, psychologic and environmental stressors. The placenta can undergo major structural and functional adaptations to shield the fetus from environmental stressors [99][100][101] . Genome-wide placental transcriptome studies have correlated gene modules involved in immune response, myeloid cell differentiation, and placental tissue development with newborn birth weight 102 . The placenta is genetically identical with the fetus 103,104 , and epidemiological studies suggest that genetic factors account for 30-80% of birth weight variance [105][106][107] . Interestingly, placental DNA methylation may be influenced by maternal insulin levels during pregnancy 108 , and increased placental DNA methylation is associated with large-for-gestational-age infants 109 , and methylation alternations may reflect changes to placental texture 110 . Nevertheless, the mechanisms underlying these observations need further interrogation.

Strengths and limitations
Our strengths include a novel, non-invasive approach to analyze in vivo placental morphometry and texture to detect early alterations in pregnant women affected by the COVID-19 pandemic mediated, at least in part, by maternal mental distress. We utilized the 3-D reconstructed models and computational analyses of texture based on high-resolution MR images to detect abnormalities, representing gross and microstructural changes in placental development during the pandemic 111,112 . The benefits of MRI lead to finer 3-D placental models and better estimation of the morphometric and textural features of human placentas presented in our study.
Although our study had several strengths, the study limitations need to be outlined. First, the segmentation of the placentas from MR images was performed manually. Due to the variability of the placental shape, orientation and appearance, the fully automatic segmentation of the placenta remains a challenge 46,113 . Thus, manual segmentation is currently the best method used in the literature and has served as the ground truth when developing segmentation algorithms for the human placenta 20,114,115 . Second, this work was intended to explore the impact of the pandemic on placental development and included women with no known COVID-19 exposures, based on serial questionnaires. However, subclinical or unknown exposures to the COVID-19 virus cannot be fully excluded. Similarly, we investigated the role of pandemic-related maternal mental distress associated with COVID-19 on placental development; however, there may be additional stressors and lifestyle changes related to the pandemic, including environmental stressors, and changes in physical activity, that also may have contributed to disrupted placental development. Furthermore, pregnant women recruited into this study were from the Washington, DC metropolitan area and the associations observed in this study should be explored in other geographic regions before assessing the generalizability of these findings. Third, the clinical implications www.nature.com/scientificreports/ of altered placental morphometry and texture on child neurodevelopmental for pregnancy during pandemic remain uncertain; ongoing studies of pregnancy outcome are currently underway. These include correlating of in vivo findings with ex vivo placental pathology, as well as long-term neurodevelopmental outcomes of children born during the pandemic. Lastly, application of these analyses and results should be linked to clinical evaluation and interpretation, in order to develop novel prediction models of in vivo placental structure relative to clinically significant outcomes [116][117][118] .

Conclusion
Our study is the first to describe MRI-based morphometric and textural changes of in vivo placenta in women pregnant during the COVID-19 pandemic. Our approach provides a semiautomated method of standardizing placental MR evaluation in women during and prior to the pandemic. With these sophisticated techniques, we can detect in vivo changes of the placenta not only on gross-level evaluation (i.e., morphometry) but also microscopic-level assessment (i.e., texture). Moreover, we demonstrate changes in gross and microscopic placental structure in women with increased levels of prenatal stress, a potentially modifiable risk factor that, if recognized early, may allow for timely interventions to improve placental health and pregnancy outcomes. Future studies relating in vivo measures of placental development with clinical evaluations and outcomes may lead to the development of clinically relevant prediction models of placental health [116][117][118] . The further evaluation of these findings with placental pathology, pregnancy outcomes and long-term neurodevelopmental health is currently under investigation.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.